Differentiation of HL-60 leukemia by type I regulatory subunit antisense oligodeoxynucleotide of cAMP-dependent protein kinase.

Medical Sciences

Differentiation

of HL-60

leukemia by type I

regulatory subunit

antisense

oligodeoxynucleotide

of cAMP-dependent protein kinase

(cAMP kinase)

GIAMPAOLO

TORTORA,

HIROSHI

YOKOZAKI,

STEFANO

PEPE,

TIMOTHY

CLAIR,

AND

YOON S.

CHO-CHUNG*

CellularBiochemistrySection, Laboratoryof TumorImmunology and Biology, National Cancer Institute, Bethesda, MD 20892

Communicated by VanRensselaer Potter, November 19, 1990(receivedforreviewOctober 5, 1989) ABSTRACT A marked decrease in the

type

I

cAMP-dependent protein kinase regulatory subunit (RIe) and an increase in the type IIprotein kinase regulatory subunit

(Rllp)

correlate withgrowth inhibition and differentiation induced in avariety of typesofhumancancer cells, in vitro and invivo, by site-selective cAMP analogs. To directly determine whether

RI,,

is agrowth-inducing protein essentialfor neoplastic cell growth, human HL-60 promyelocytic leukemia cells were exposed to 21-mer RIa antisenseoligodeoxynucleotide, andthe effects on cell replicationand differentiation were examined. The

RI,

antisense oligomerbrought about growthinhibition and monocytic differentiation, bypassing the effects of an exogenous cAMP analog. These effects of

RI.

antisense oli-godeoxynucleotidecorrelated with a decrease in

RI.

receptor andanincrease in

Rllp

receptor level. Thegrowthinhibition and differentiation wereabolished, however,when thesecells wereexposed simultaneously to both

RI.

and

RUp

antisense oligodeoxynucleotides. The

RIIp

antisense

oligodeoxynucleo-tide alone has beenpreviouslyshown tospecificallyblock the differentiation induciblebycAMPanalogs. These results pro-vide direct epro-vidence that

RI.

cAMP receptorplays a critical role inneoplasticcellgrowth and that cAMP receptorisoforms displayspecific roles incAMPregulation of cell growth and differentiation.

cAMP-dependent

protein

kinase is composed oftwo genet-icallydistinctsubunits, catalytic (C) and regulatory (R). The activating ligand cAMP, which binds to the R, induces conformational changes and dissociates holoenzyme R2C2 intoanR2(cAMP)4dimer andtwofree C thatarecatalytically active (1, 2). There are two different classes of cAMP-dependent protein kinase designated as type I and type

II,

which contain distinct R,RIandRII,respectively, but share a commonC (3, 4). Fourdifferent regulatory subunits

[RI,

(RI) (5),

RIp

(6),

RIIa,,

(RI154) (7), and

R13

(RI151) (8)] have beenidentified atthe

gene/mRNA

level.Twodistinct C

[C,

(9) and

Cp

(10, 11)] have also been identified; however, preferential coexpression of eitheroneof theseC with either the type I or type IIprotein kinaseRhasnotbeen found(11). It has been shown that the ratio of cAMP-dependent protein kinases type Iandtype II variesamongtissues and thatan increased

expression

of

protein

kinasetype I orRI correlates with active cell growth, cell transformation, or early stages of differentiation(12).

We

hypothesized

that RIisanontogenic growth-inducing protein,and its constitutive expression disrupts normal on-togenicprocesses, resulting inapathogenicoutgrowth, such asmalignancy. Todirectlydetermine whether RI has a role in neoplastic cell growth, at least in vitro, we used the antisense strategy (13). We used 21-mer synthetic

oligode-oxynucleotides

complementary to either the sense or

an-tisense strands of the human RIa (14) mRNA

transcript

starting from the first codon (15-17). We thenexposed human HL-60 leukemia cells to these

oligodeoxynucleotides

and examined the effects of this treatment on cell

proliferation

anddifferentiation. Asadditional controls, we also

synthe-sized RIIa (18) and

RIIp

(19) antisense and a random-sequence

oligodeoxynucleotide

of identical size.

MATERIALS AND

METHODS

Oligodeoxynucleotides. The21-mer

oligodeoxynucleotides

used in the present studies were synthesized at Midland Certified Reagent

(Midland,

TX) and had the

following

se-quences: human RIa (14) antisense, 5'-GGC-GGT-ACT-GCC-AGA-CTC-CAT-3'; human

RII,

(19) antisense, 5'-CGC-CGG-GAT-CTC-GAT-GCT-CAT-3'; human RIla (18)

antisense,

5'-CGG-GAT-CTG-GAT-GTG-GCT-CAT-3';

and the random-sequence

oligodeoxynucleotide

was made ofa mixture of all four nucleotides atevery

position.

Cel

Growth

Experiment.

Cellsgrownin

suspension

culture in RPMI 1640

medium/l0o

fetal bovine

serum/penicillin

at 50

units/ml/streptomycin

at500

,ug/ml/1

mM

glutamine

(GIBCO)

were seeded at 5 x

105

cellsper dish. Oligodeoxynucleotides wereaddedafter seeding andevery48 hrthereafter. Cellcounts were performed on a Coulter Counter. Cells unexposed or exposed to oligodeoxynucleotides for 4 days were reseeded (day 0)at5 x

105

cells perdish, and cells preexposed tothe

oligodeoxynucleotide

werefurther treated with the

oligomer

at

day

0 andday 2. cAMP analogs (provided by R. K.

Robins,

Nucleic Acid Research Institute, CostaMesa,CA)or

phorbol

12-myristate 13-acetate (PMA)wereaddedonetimeatday 0. Cellcountswere

performed

onday4.

Immunoprecipitation

of

RIa

and

Rllp

cAMP Receptor Pro-teinsAfter

Photoaflnmity Labeling

with8-Azidoadenosine

3',5'-Cyclic

[32P]Monophosphate

([32P18-N3-cAMP).

Cell extracts werepreparedat

0-40C.

Thecell pellets(2x

106

cells),after two washeswith

phosphate-buffered

saline, were

suspended

in0.5 mlof bufferTen(0.1 MNaCl/5

mM

MgCl2/1% Nonidet

P40/0.5%

sodium

deoxycholate/bovine

aprotinin at 2 kal-likrein inhibitor units per

ml/20 mM Tris-HCl,

pH 7.4) con-taining proteolysis inhibitors (17), Vortex-mixed, passed througha22-gauge needle10times, allowedtostandfor 30 min at

40C,

and centrifuged at750 x gfor20 min; the resulting supernatants were used as cell

lysates.

The photoactivated incorporation of

[32P]8-N3-cAMP

(60.0 Ci/mmol; 1 Ci = 37 GBq) and the immunoprecipitation using the

anti-RI,,,

or

anti-RII,6

antiserum(provided by S.

0. D0skeland,

University of Abbreviations: C, catalytic subunit(s); R, regulatory subunit(s); PMA, phorbol 12-myristate13-acetate; RIand RII,type Iandtype

lI R, respectively;

[32P]8-N3-cAMP,

8-azidoadenosine 3',5'-cyclic

[32P]monophosphate;

8-Cl-cAMP, 8-chloroadenosine 3',5'-cyclic monophosphate.

*Towhomreprintrequestsshould beaddressedat:NationalCancer Institute, National Institutes ofHealth, Building 10, Room5B38, Bethesda,MD20892.

2011 Thepublicationcostsof this articleweredefrayedinpartbypagecharge

payment. This article must therefore be hereby marked "advertisement" inaccordancewith 18U.S.C. §1734 solely to indicate this fact.

2012 Medical Sciences: Tortora et al.

Bergen, Bergen, Norway) and protein A-Sepharose andSDS/ PAGE ofsolubilized antigen-antibodycomplex followed the method described (17, 20).

cAMP-DependentProtein Kinase Assays. After twowashes with Dulbecco'sphosphate-buffered saline, cell pellets (2 x 106 cells) were lysed in 0.5 ml of 20 mM Tris, pH 7.5/0.1 mM sodium EDTA/1 mM dithiothreitol/0.1 mM pepstatin/0.1 mM antipain/0.1 mM chymostatin/0.2 mM

leupeptin/

aprotininat 0.4mg/ml/soybeantrypsin inhibitor at 0.5mg/ml, using100strokes ofaDounce homogenizer. After centrifu-gation (Eppendorf5412) for 5 min, the supernatants were adjusted to 0.7 mg of protein per ml and assayed (21) immediately.Assays

(40,ul,

totalvolume) wereperformedfor 10 min at 30'C and contained200

,4M

ATP, 2.7 x 106 cpm [_y-32P]ATP,20 mMMgCl2,

100,/M

kemptide (Sigma K-1127) (22), 40 mMTris(pH 7.5), ± 100

,uM

protein kinase inhibitor (Sigma P-3294) (23), ±8,uMcAMPand7

,ug

of cell extract. Thephosphorylation of kemptidewasdeterminedbyspotting 20 ,l of incubation mixture on phosphocellulose filters (Whatman P81) and washing them in phosphoric acid as described (24). Radioactivity was measured by liquid scin-tillation with Econofluor-2 (NEN ResearchProducts NEF-969).

Isolation of Total RNA and Northern (RNA) Blot Analysis. Thecells

(108

washedtwice withphosphate-buffered saline) werelysedin 4.2 Mguanidineisothiocyanate/25mMsodium citrate, pH7.0/0.5% sarcosyl (N-lauroylsarcosine

Na+)/0.1

M 2-mercaptoethanol, and the lysates were homogenized; total cellularRNA wassedimented through aCsClcushion (5.7 MCsCl/10mM EDTA)asdescribedby Chirgwinetal. (25).Total cellularRNA in 20 mM

3-(N-morpholine)propane-sulfonicacid, pH

7.0/50%

(vol/vol)

formamide/6%

(vol/vol)

formaldehyde wasdenaturedat65°C for10minand electro-phoresed through adenaturing 1.2% agarose/2.2 M formal-dehydegel.Thegelswerethentransferred to Biotransnylon membranes(ICN) bythemethod ofThomas(26)and hybrid-ized to thefollowing two32P-labeled nick-translated cDNA probes: 1.5-kilobase (kb) cDNA clone containing the entire codingregion forthe humancAMP-dependent

protein

kinase type I R, RIa (14)

(provided

by T. Jahnsen, Institute of Pathology,Rikshospitalet, Oslo, Norway)and human 8actin (Oncor p70001 actin).

RESULTS

AND

DISCUSSION

The

RIa

antisense

oligodeoxynucleotide

at 15 ,uM concen-tration(17) hadimmediate effectsontherateof

proliferation

ofHL-60cells.By4-5days inculture,cellsunexposedtoRIa antisense oligomer demonstrated an exponential rate of growth,whereascells

exposed

totheRIa antisense

oligomer

exhibited a reduced

growth

rate and

eventually

stopped

replicating (Fig. 1A). This

inhibitory

effectoncell

prolifera-tion

persisted throughout

the culture

period.

The

growth

inhibitionwas notduetocell

killing;

cellswere>90%viable afterexposure to

RIa

antisense

oligomer (15

,uM)

for7

days

as assessed by flow cytometry by

using

forward and side scatter.

RIa,

sense,

Rlla,

RIIf3

antisense,

or a random-sequence

oligodeoxynucleotide

had no such

growth

inhibi-toryeffect.

Cellsunexposed orexposedtoRIa antisense

oligodeoxy-nucleotide for4daysinculturewerereseeded andexamined for theirresponsetotreatmentwith cAMPanalogsorPMA. In cells unexposed to RIa antisense

oligodeoxynucleotide,

8-chloradenosine

3',5'-cyclic

monophosphate

(8-Cl-cAMP)

(10

,uM)

produced

60%

growth inhibition, and 80% growth inhibition was achieved by 8-Cl-cAMP (5 ,uM) plus

N6-benzyl-cAMP(5,uM)(Fig. 1B) (27); PMA(10-8M)exhibited 60%growth inhibition

(Fig.

1B). In contrast,cells

exposed

to RIa antisense

oligodeoxynucleotide

exhibited retarded growth

(=25%

the rate ofgrowth ofcells

unexposed

tothe

A

3 0 ox C.)0o x E z 2 1

B

en 0 0 L-. E z (-)Rla antisonse (+) RI,> antisense 2 4 6 8 TIME (days) (-)

RIs

(+)RIa antisense antisense " 88P C 88P CICIM CICIM +A +A N6 N6 B B

FIG. 1. Effect ofRIaantisenseoligodeoxynucleotideonthebasal

rate ofgrowthof HL-60 leukemic cells(A) and thegrowthof these cells when treated with cAMPanalogsorPMA (B). (A) Cellswere

grownwithout(o)orwith

(e)

RI,,

antisenseoligodeoxynucleotide(15

AM).

Atindicated times, cellcountsin duplicatewere performed.

Datarepresentthe average values±SDs of fourexperiments. (B)On day4ofexperimentA, cellsexposedorunexposedtoRlaantisense oligodeoxynucleotidewerereseeded(day 0)at5x 105cells perdish, and cells preexposed to RIaantisenseoligodeoxynucleotide were

furthertreatedwith theoligomeratday0and 2. cAMPanalogsand

PMA were added once atday0. Cells were countedon a Coulter Counter on day 4. 8-Cl, 8-Cl-cAMP (10

AM);

8-Cl +

N6-B,

8-Cl-cAMP (5 ,M) +

N6-benzyl-cAMP

(5 ,uM); PMA

(10-8

M). Data represent the average values ± SDs of fourexperiments.

RIa

antisense

oligomer),

andneither cAMP

analogs

norPMA furtherretarded

growth (Fig. 1B).

HL-60 cells

undergo

a

monocytic differentiation

upon treatmentwithsite-selective cAMP

analogs

(27).

Cells either

unexposed

or

exposed

toRIaantisense

oligodeoxynucleotide

wereexamined for their

morphology

before and after treat-ment with cAMP

analogs.

As shown in

Fig.

2,

in cells

unexposed

to RIa antisense

oligomer, 8-Cl-cAMP

plus

N6-benzyl-cAMP

induced a

monocytic

morphologic

change

characterized

by

a decrease in

nuclear-to-cytoplasm ratio,

abundant ruffled and vacuolated

cytoplasm,

and loss of nucleoli

(27).

Strikingly,

the same

morphologic

change

was

inducedwhen cells were

exposed

to RIa antisense

oligode-oxynucleotide (Fig.

2). Moreover,

the

morphologic

changes

induced

by RIa

antisense

oligomer

were

indistinguishable

from that induced

by

PMA

(Fig.

2).

To

provide

more evidence that the

growth

inhibition and

monocytic

differentiation inducedin HL-60cells

exposed

to

the

RI,

antisense

oligodeoxynucleotide

were due to an in-tracellular effect of the

oligomer,

we determined the RIa mRNA level. As shown in

Fig.

3,

3.0-kb

RI,

mRNA

(14)

was

virtually

undetectable in cells

exposed

for 8 hr to RIa

an-tisense

oligodeoxynucleotide (Fig.

3B,

lane

2),

and the de-creaseinRIamRNAwasnotdueto aloweramountof total RNA asshown

by

the ethidium bromide

staining

(compare

lane 2 with lane 1of

Fig.

3A).

Conversely,

anenhanced level of actinmRNAwasdetectedincells

exposed

to

RI,

antisense

oligomer

(Fig.

3B).

Whether the increase inactinmRNAlevel represents

changes

in

cytoskeletal

structure isnotknown.

We nextdeterminedthelevelsof cAMPreceptor

proteins

in these cells

by

immunoprecipitation

with anti-RIa and Proc. Natl. Acad. Sci. USA88

(1991)

Proc. Natl. Acad. Sci. USA 88 (1991) 2013

-

RI,,

Antisense

+

RI..

Antisense

4,~~~

&

Conrol

04'

0a 8-Cl-cAMP 6 N

-Benzyl

j cAMP

PMA

<

o

And

@

9~~~~~~~~~~~~~~~~~~~~~~~

0

A_

S

* FIG. 2. Effect of

RI,,

antisense

oh-godeoxynucleotide on the morphologic

transformation of HL-60 cells. Cells

ei-ther exposed or unexposed to RI<,

an-tisense oligodeoxynucleotide were

treated with cAMP analogsor PMAas

described in Fig. 1B. On day4(seeFig. 1B),cellswerewashedtwice in

Dulbec-co'sphosphate-buffered saline andwere

pelletedontoaglass slide by

cytocentri-fuge. The resulting cytopreparations

werefixed and stained by Wright's stain.

(x120.)

anti-RII#

antisera (17, 20) after photoaffinity labeling of these

receptor proteins with [32P]8-N3-cAMP. In control cells,

treatment with 8-Cl-cAMP plus NM-benzyl-cAMP reduced RIa -70% and increased

Rh1p

3-fold, resulting in a 10-fold

increase in theratio of RII13/RIh (Fig. 4) (28). Exposure of these cellstoRIa antisense oligodeoxynucleotide for 4 days brought about marked changes in bothRIaandRh11 levels;an

80%reduction inRIa witha5-fold increase in Rh11 resulted

ina25-fold increase in the ratio of RII1/Rla compared with

that in control cells (Fig. 4). Because growth inhibition and differentiationwereappreciable after 3-to4-dayexposureto

RIaantisense oligomer, the changing levels of RIa and RIIS

A

RLI antisense

B

RI0 antisense 28S 8S M 1 2 Rh0 -3.0 Kb Actin -2.2 Kb 1 2

FIG. 3. Decreased RI, mRNA expression in HL-60 leukemic

cells exposed to RIa antisense oligodeoxynucleotide. Cells were

eitherexposedorunexposedtoRI, antisenseoligodeoxynucleotide (15

AM)

for 8 hr. Isolation of total RNA andNorthern blotanalysis

followed thedescribed methods. (A)Ethidium bromide stainingof RNA.Lanes:M,markers of ribosomalRNAs; 1,cellsunexposedto

RI, antisenseoligomer; 2,cellsexposedtoRI,, antisenseoligomer. (B)Northern blotanalysis;thesamenitrocellulose filterwas

hybrid-izedtobothRIaand actin probesinsequentialmanner. Lanes: 1,

cellsunexposed toRIa antisenseoligomer; 2,cellsexposed toRIa antisenseoligomer.

proteinsappearanearlyeventnecessaryfor commitmentto

differentiation.

Data in Fig. 4 showed that suppression of RIa by the antisense oligodeoxynucleotide brought about a

compensa-toryincrease in

RMgS

level. Such coordinated expression of RI andRII withoutchanges in theamountof C has been shown (29, 30). The increase in Rh13 may be responsible for the

differentiation induced in these cells after exposure to RIa antisenseoligodeoxynucleotide. The increase in RIIh mRNA

orRh13 protein level has been correlated with cAMP

analog-induceddifferentiation in K-562 chronic myelocytic leukemic cells(31) and in erythroid differentiation of Friend erythro-cytic leukemic cells (32). In a recent report (17), we have

provided direct evidence thatRIIh is essential for the cAMP-induceddifferentiation inHL-60 cells. HL-60 cells thatwere

exposedtoRh13 antisense oligodeoxynucleotide became

re-fractory to treatmentwith cAMP analogs and continued to

grow.

The essential role of RII3 in differentiation of HL-60 cells

wasfurtherdemonstrated when these cellswereexposedto

both

RI,

andRh11 antisenseoligodeoxynucleotides simulta-neously. As shown in Table 1,

RI,,

antisense oligodeoxynu-cleotide markedly increased the expression of monocytic surfaceantigens [Leu 15 (33) and Leu M3 (34)] and decreased markers relatedtothe immaturemyelogenous cells [My9 (35, 36)]. These changes in surface marker expression were

abolished when cells were exposed simultaneously to both RIaandRII3 antisense oligodeoxynucleotides (Table 1). RIIa cAMPreceptorwasnotdetected in HL-60 cells(37), and RIa antisense oligodeoxynucleotide showednointerference with

theeffects of

RI,,,

antisense oligomer (Table 1).

Cellsexposedtoboth

RI,

and Rh13 antisense oligodeoxy-nucleotideswere neithergrowth inhibitednordifferentiated

regardless of cAMP analog treatment. We interpret these results to reflect the blockage of cAMP-dependent growth regulatory pathway. Cells under these conditions are no

longer cAMP-dependent but survive and proliferate probably throughanalternatepathway. Thus, suppression of both RIa

.s

Medical Sciences: Tortoraetal.

tt, *

I

v

2014 Medical Sciences: Tortoraetal.

A

al 68K W RI,, intisense B RIL, antisense 43K *4 -29K

M 1 2 3 RI,,RIL, M 1 2 3R1RII,,

Relative Levels

Lane Treatment

RI,,

REH

1 Control 1.0±wt0.1 1.0+0.1

2 8-Cl +

N6-Benzyl

0.3± 0.04 3.0±0.3

3 RI, Antisense 0.2±0.03 5.0±0.4

FIG.4. Effect ofRI, antisenseoligodeoxynucleotideonthe basal

and inducedlevels of RIaandRIH, cAMPreceptorproteins in HL-60 leukemic cells. CellswereeitherexposedtoRIaantisense oligode-oxynucleotide (15 MM)ortreated withcAMPanalogsasdescribed in

Fig.1.Preparationofcellextracts,thephotoactivated incorporation of [32P]8-N3-cAMP and immunoprecipitation with the anti-RIa or

anti-RIIp

antiserum and protein A-Sepharose, and SDS/PAGE of

solubilized antigen-antibody complex followed the methods de-scribed. Preimmuneserumcontrolswerecarriedoutsimultaneously and showed no immunoprecipitated band. Lanes: M, 14C-labeled

marker proteins of known Mr; RIa, 48,000 Mr RI (Sigma); RIIa, 56,000 MrRII (Sigma). Lanes RI, andROILarefromphotoaffinity labelingwith[32P]8-N3-cAMP only; lanes 1-3, photoaffinity labeling with[32P]8-N3-cAMP followed by immunoprecipitation with

anti-RI,

or anti-RI1 antiserum. 8-Cl, 8-Cl-cAMP (5 uM); N6-benzyl,

N6-benzyl-cAMP (5 uM). Data in the tablerepresentquantification by densitometricscanningofautoradiograms. Dataareexpressed

rel-ativetolevelsin control cells unexposedtoRIa antisenseoligomer

and untreated with cAMPanalog,whicharesetequaltoonearbitrary

unit. Datarepresentanaverage± SD of threeexperiments.

Immu-noprecipitation waswith anti-RIa(A)or

anti-Rllp3

(B)antisera.

andRIIh geneexpression ledtoanabnormal cellulargrowth

regulation similar tothat in thosemutant cell lines(38) that contain either deficient or defective regulatory subunits of cAMP-dependent proteinkinaseandare nolonger sensitive

to cAMP stimulus.

OurresultsdemonstratedthatcAMPtransducessignalsfor dual, either positiveor negative, controls oncell

prolifera-tion, depending on the availability of

RI,

or REII receptor

Table 1. Modulation of differentiation markers in HL-60 cells by RI, antisense oligodeoxynucleotide

Surface markers, % positive

Treatment Leu 15 Leu M3 My9

Control 10 2 100

RI, antisense 80 98 80

RI,

antisense + RIIM antisense 11 2 100

RIIp

antisense 13 3 100

RI,

antisense +

RII.

antisense 85 100 80 Surface-antigens were analyzed by flow cytometry and monoclo-nal antibodies (Leu 15, Leu M3, and My9) reactive with either monocytic or myeloid cells. Approximately 2 x 104 cells were analyzed for each sample, and cells weregated by using forward and side scatter. Numbers represent the average values of three exper-iments.

proteins. The RIa antisense oligodeoxynucleotide, which suppressed RIaand enhancedRIIB expression, ledto termi-naldifferentiation of HL-60 leukemia with no sign of

cyto-toxicity.

It is unlikely that free C increase in cells exposed to RIa antisense oligodeoxynucleotide wasresponsible for the

dif-ferentiation because cells exposed toRIIB antisenseorboth

RI,

and RIIB antisense oligodeoxynucleotides, conditions that also would produce free C, continued to grow and

became refractorytocAMP stimulus. To directly verify this

we measured phosphotransferase activity in cells that are

exposed or unexposed to the antisense oligodeoxynucleo-tidesusing Kemptide (22)asasubstrate in thepresenceand

absence ofa saturating concentration of cAMP and in the presence and absence of the heat-stable protein kinase in-hibitor(23). This method ofassaygivesaccurate determina-tion of the relativelevels of dissociated C and total C activity. Cellextractsfromuntreated HL-60 cells exhibitedaverylow levelof dissociated C andwerestimulated 36-fold by cAMP

(Table 2). This cAMP-stimulated activity was almost

com-pletely inhibited by the heat-stable protein kinase inhibitor (Table 2), indicating that the total C activity measuredwas

cAMP-dependent protein kinase. In cells exposed to RIa antisense, RIIB antisense,orRIaand

Rh,3

antisense oligode-oxynucleotides, the free C activity was not increased as

compared with unexposed control cells, although the total cAMP-stimulated activity differed slightly (Table 2). These resultsprovide direct evidence that free C isnotresponsible for the differentiation seenin HL-60 cells.

Overexpression ofRIa cAMP receptor protein has also been found in most human breast and colonprimary

carci-nomasexamined(39), suggestinganimportant in vivo role of

Table 2. Protein kinaseactivityin HL-60 cells

-cAMP +cAMP

Relative Relative Stimulation,

Treatment Activity* tocontrol Activity* tocontrol -fold

-PKI

Control 23.0± 6.6 1.0 837 ± 87 1.0 36

RIa antisense 22.9± 5.4 1.0 944 ± 18 1.1 41

RMIg

antisense 22.8± 8.1 1.0 1028 ± 154 1.2 45

RIa and

RMg

antisense 24.3± 7.0 1.1 802 ± 36 1.0 33

+PKI

Control 17.5 ± 8.7 1.0 37.0 ± 8.4 1.0 2.1

RIa antisense 25.0± 8.8 1.4 22.6 ± 8.8 0.6 0.9

RIIp

antisense 24.0 ± 2.6 1.4 24.8 ± 3.9 0.7 1.0

RIa and ROIL antisense 19.0 ± 5.9 1.1 19.1 ± 8.2 0.5 1.0

Cellswereexposedto 15MMconcentrations ofRI,,

RMIg,

or to15 uM each of both antisenseoligodeoxynucleotidefor

4days(seeFig. 1A). Data representanaverage ±SD ofduplicatedeterminations from three identicalexperiments.PKI, proteinkinase inhibitor.

*pmol of phosphate transferredtokemptidepermin/mgofprotein.

RIa cAMPreceptorin tumorgrowth aswell. However, the

precise role of

RI,

incellproliferationisnot yetknown. RIa

may suppressRII production by titratingoutC,oritmaybe atransducerofmitogenic signals leadingtocellproliferation. Our results show that

RI,

antisense oligodeoxynucleotide providesauseful genetic tool for studiesonthe role of cAMP

receptorproteinsincell proliferation and differentiation and contributetoanotherapproachinthe control ofmalignancy.

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